miR-92a在脑胶质瘤和脑胶质瘤干细胞中的作用研究

发布时间：2018-07-10 11:15

本文选题：miR-92a + 脑胶质瘤　；参考：《东南大学》2017年博士论文

【摘要】：脑胶质瘤是中枢神经系统中最常见的原发性肿瘤之一,恶性水平高,具有很高的发病率与死亡率。近年来,虽然人们在脑胶质瘤的手术医治以及术后放疗、化疗等方面取得了长足的进步,但是恶性脑胶质瘤病人的预后依然很差,确诊后的平均生存周期不超过15个月。最新的研究发现,脑胶质瘤细胞中存在一小部分具备干细胞特征的细胞亚群,这类细胞可以持续增殖,具备自我更新能力和多向分化潜能,被定义为脑胶质瘤干细胞(Glioma stem cells, GSCs)。GSCs细胞的存在是脑胶质瘤形成、浸润、转移、复发以及放化疗耐受的主要原因。MicroRNAs (miRNAs)是一类在进化上高度保守,长度大约在22nt左右的非编码RNA分子,可以通过直接作用于下游mRNAs的3端非翻译区(3'Untranslated Regions,3'UTR)阻止mRNAs的转录,和域诱导mRNAs的降解,进而在转录后水平调控机体内基因的表达。越来越多的证据表明,miRNAs分子能够起着类似于原癌基因或者抑癌基因的作用,影响肿瘤细胞的形成与发展。此外,也有文献报道指出miRNAs在肿瘤干细胞分化和自我更新的调控中也发挥着重要的功能。因此,可以通过改变miRNAs的表达量来降低肿瘤细胞和肿瘤干细胞的恶性程度,这可能是一个很有前景的治疗方案。在本研究中,作者通过高通量测序技术筛查发现,与脑胶质瘤细胞相比,GSCs细胞中miR-92a的表达水平显著降低。之前的文献报道表明,miR-92a在脑胶质瘤细胞系和脑胶质瘤病人的组织标本中高表达,降低miR-92a的表达水平可以抑制脑胶质瘤细胞的增殖并促进细胞凋亡。然而,miR-92a在脑胶质瘤细胞迁移、侵袭以及GSCs细胞生存、迁移和自我更新中的功能尚不明确。所以,充分了解miR-92a在这两种细胞中的功能和具体分子机制,可以为脑胶质瘤的治疗提供新的思路。研究目的探索miR-92a在脑胶质瘤以及GSCs细胞中的功能和详细作用机制,为脑胶质瘤的靶向治疗提供新的理论依据。实验方法1.采用无血清饥饿培养法从脑胶质瘤细胞(U87和U251)中分离培养出GSCs细胞,并用免疫荧光染色法鉴定GSCs细胞;2.采用第二代高通量测序技术比较U87细胞和来源于U87的GSCs细胞U87s中miRNAs的表达量差异;3.采用MTT、细胞划痕、Transwell以及Annexin V-FITC/PI双染法检测miR-92a对U87和U251细胞体外增殖、迁移、侵袭和凋亡的影响;4.采用裸鼠成瘤实验检测miR-92a在体内对脑胶质瘤细胞生长的影响;5.采用qPCR、Western Blot和肿瘤干细胞球成球等实验检测miR-92a对GSCs细胞生长、迁移以及自我更新的影响;6.结合生物信息学方法和双荧光素酶报告系统预测并鉴定miR-92a在脑胶质瘤细胞和GSCs细胞中的靶基因;7.采用Western Blot和免疫荧光染色法确定miR-92a在脑胶质瘤细胞和GSCs细胞中作用的分子机制。实验结果1.利用无血清饥饿培养法从脑胶质瘤细胞(U87和U251)中分离出GSCs细胞(U87s和U251s),免疫荧光染色实验结果表明,GSCs细胞表面表达肿瘤干细胞的分子标志CD133和神经干细胞的分子标志Nestin,分化后的GSCs细胞表达神经胶质细胞的分子标志GFAP以及神经元细胞的分子标志β-tubulin Ⅲ;2.高通量测序的分析结果显示共有94个miRNAs在U87细胞和U87s细胞间差异表达,其中miR-92a在U87s细胞中显著低表达。qPCR鉴定结果表明miR-92a在U87s细胞和U251s细胞中均显著下调;3.下调miR-92a的表达量在体外可以有效的降低U87细胞和U251细胞的增殖和转移能力,同时促进细胞凋亡;4.降低miR-92a的表达水平可以有效的抑制脑胶质瘤裸鼠模型体内肿瘤的生长;5.在GSCs细胞中上调miR-92a的表达量后,肿瘤干细胞球直径变小、细胞迁移能力减弱、肿瘤干细胞球形成率降低,细胞干性维持相关分子标志表达量下调;6.生物信息学预测和双荧光素酶报告系统结果表明miR-92a在脑胶质瘤细胞和GSCs细胞中可作用于不同的靶基因,miR-92a在脑胶质瘤细胞中的靶基因为CDH1,在GSCs细胞中的靶基因则为Notch-1;7.脑胶质瘤细胞内miR-92a的表达量下调后,CDH1蛋白量增多,细胞核内p-catenin含量减少,而总的p-catenin含量不变;8.在GSCs细胞内过表达miR-92a后,Notch-M蛋白下调,细胞内磷酸化Akt的含量减少,而总的Akt含量不变;9.过表达CDH1可以抑制脑胶质瘤细胞的转移能力,而降低Notch-1的表达则可抑制GSCs细胞的生长、迁移和自我更新能力。结论本研究首先从脑胶质瘤细胞中分离出GSCs细胞,通过高通量测序技术发现miR-92a在两种类型的细胞中差异表达。qPCR结果证实,与脑胶质瘤细胞相比,GSCs细胞内miR-92a的含量明显降低。降低miR-92a的表达水平可有效抑制脑胶质瘤细胞的增殖、迁移、侵袭,并促进细胞凋亡;miR-92a含量减少后,脑胶质瘤裸鼠模型体内肿瘤生长速度也明显减慢。另一方面,上调miR-92a的含量可以显著抑制GSCs细胞的生长,迁移和自我更新。深入的机制研究表明,miR-92a可以通过分别作用于下游CDHl/p-catenin和Notch-1/Akt信号通路,影响脑胶质瘤细胞和GSCs细胞的恶性表型。上述实验结果表明miR-92a在脑胶质瘤的形成和发展过程中起重要作用。
[Abstract]:Glioma is one of the most common primary tumors of the central nervous system with high malignant level and high morbidity and mortality. In recent years, although people have made considerable progress in surgical treatment of glioma, radiotherapy and chemotherapy, the prognosis of patients with malignant glioma is still poor and after diagnosis. The average life cycle is not more than 15 months. The latest research has found a small group of cell subgroups with stem cell characteristics in glioma cells, which can continue to proliferate, have self-renewal ability and multidirectional differentiation potential, and are defined as brain glioma stem cells (Glioma stem cells, GSCs).GSCs cells are brain glue The main cause of tumor formation, infiltration, metastasis, recurrence and chemotherapy tolerance.MicroRNAs (miRNAs) is a class of non coded RNA molecules, which are highly conserved in evolution and are about 22nt around 22nt, and can prevent mRNAs's transcription by directly acting on the 3 terminal non translation region (3'Untranslated Regions, 3'UTR) downstream of the downstream mRNAs, and the domain induces mRNAs. There is a growing number of evidence that miRNAs molecules can play a role similar to proto oncogenes or tumor suppressor genes to affect the formation and development of tumor cells. In addition, there are also reports that miRNAs also plays a role in the regulation of differentiation and self renewal of cancer stem cells. Therefore, it is possible to reduce the malignancy of tumor cells and tumor stem cells by changing the expression of miRNAs, which may be a promising treatment. In this study, the authors found that the level of miR-92a expression in GSCs cells decreased significantly compared with glioma cells. Low. Previous reports suggest that miR-92a is highly expressed in the tissue specimens of glioma cell lines and glioma patients. Reducing the expression level of miR-92a can inhibit the proliferation of glioma cells and promote cell apoptosis. However, miR-92a migration, invasion, and GSCs cell survival, migration and self renewal in glioma cells. The function and specific molecular mechanism of miR-92a in these two cells can provide new ideas for the treatment of glioma. The purpose of this study is to explore the function and detailed mechanism of miR-92a in glioma and GSCs cells, and to provide a new theoretical basis for the targeting therapy of glioma. Methods 1. the GSCs cells were isolated and cultured from glioma cells (U87 and U251) by serum-free starvation, and GSCs cells were identified by immunofluorescence staining. 2. the difference of miRNAs expression in U87 cells and U87 derived GSCs cells U87s was compared with second generation high-throughput sequencing technology; 3. used MTT, cell scratch, Transwell, and An. Nexin V-FITC/PI double staining was used to detect the effect of miR-92a on the proliferation, migration, invasion and apoptosis of U87 and U251 cells in vitro. 4. the effect of miR-92a on the growth of glioma cells in vivo was detected by nude mice, and the growth, migration and self of miR-92a to GSCs cells were detected by qPCR, Western Blot and tumor stem cells. The effect of update; 6. combined with bioinformatics and double luciferase reporting system to predict and identify the target genes of miR-92a in brain glioma cells and GSCs cells; 7. the molecular mechanism of miR-92a in glioma and GSCs cells was determined by Western Blot and immunofluorescence staining. Experimental results 1. use serum-free starvation. GSCs cells (U87s and U251s) were isolated from brain glioma cells (U87 and U251). The results of immunofluorescence staining showed that GSCs cells expressed the molecular markers CD133 of tumor stem cells and molecular markers Nestin of neural stem cells, and the molecular markers of neuroglial cells expressed as GFAP and neuron cells were expressed in the differentiated GSCs cells. The molecular marker beta -tubulin III; 2. high throughput sequencing results showed a total of 94 miRNAs differentially expressed between U87 cells and U87s cells, and the significant low expression of.QPCR identification in U87s cells showed that miR-92a was significantly downregulated in U87s and U251s cells, and 3. down-regulation of miR-92a expression was effective in vitro. Reduce the proliferation and metastasis ability of U87 and U251 cells, and promote cell apoptosis. 4. reducing the expression level of miR-92a can effectively inhibit the growth of tumor in the nude mice model of glioma. 5. after the expression of miR-92a in GSCs cells, the diameter of the tumor stem cells becomes smaller, the cell migration ability is weakened, and the tumor stem cells are spherical. 6. bioinformatics prediction and double luciferase reporter system results showed that miR-92a could play a role in different target genes in brain glioma cells and GSCs cells. The target gene of miR-92a in glioma cells was CDH1, and the target gene in GSCs cells was Notch-1; 7 After the expression of miR-92a in glioma cells was down, the amount of CDH1 protein increased, the content of P-catenin in the nucleus decreased and the total P-catenin content was unchanged. 8. after overexpression of miR-92a in GSCs cells, the Notch-M protein was down, the content of phosphorylated Akt in the cell decreased and the total Akt content was unchanged; 9. overexpression CDH1 could inhibit glioma fine. The expression of Notch-1 can inhibit the growth, migration and self renewal of GSCs cells. Conclusion this study first isolated GSCs cells from brain glioma cells. Through high throughput sequencing, the differential expression of miR-92a in two types of cells was found to be confirmed by.QPCR, compared with glioma cells, GSCs The content of miR-92a in cells decreased significantly. Reducing the expression level of miR-92a could effectively inhibit the proliferation, migration, invasion and apoptosis of glioma cells. After the decrease of miR-92a, the growth rate of tumor in the nude mice model of brain glioma was also slowed down. On the other hand, up regulation of miR-92a could significantly inhibit the GSCs cells. Growth, migration and self renewal. Deep mechanism studies have shown that miR-92a can affect the malignant phenotype of glioma cells and GSCs cells by acting on the downstream CDHl/p-catenin and Notch-1/Akt signaling pathways respectively. These results suggest that miR-92a plays an important role in the formation and development of glioma.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.41

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